A general goal in optical fluorescence microscopy is to improve the resolution much below the optical limit, which is about 250 nm Recent techniques, such as STORM (Bates, et al., Science, 317, p. 1749-53, 2007) and PALM (Betzig et al., Science, 313, p. 1642-5, 2006) have achieved ˜20 nm. In general, visible fluorescence is used to track the motion or position of a biological marker of interest. But in order to get such fine resolution (˜20 nm), one must be sure that either that the stage does not undergo motion greater than this amount, or to account for this motion and then subtract it off.
The introduction of a fiducial mark or a reference point provides a means to measure and minimize objective-sample drift. For example, a micrometer-sized bead can be affixed to the microscope cover slip, the position of which is deduced using video-imaging analysis. Such analysis has been used in a feedback loop to stabilize an optical microscope in three dimensions, recently achieving ˜0.8 nm stabilization in each axis at 25 Hz (Steffen, et al. Proc. Natl. Acad. Sci. U.S.A. 98, 14949-14954 (2001); Capitanio, et al. Eur. Phys. J. B 46, 1-8 (2005). More recently, Carter et al. (Applied Optics 46, 421-27, 2007) described stabilization of an optical microscope to 0.1 nm in three dimensions by measuring the position of a fiducial mark coupled to the microscope cover slip using back-focal-plane (BFP) detection and correcting for the drift using a piezoelectric stage. See also Perkins et al., U.S. Pat. No. 7,928,409.
However, these methods are expensive, require sophisticated equipment and/or are difficult to use effectively. For example, Carter et al. (supra) requires electron beam lithography and an extremely sophisticated optical trap to control the stage in 3 dimensions. Simpler gold particles or beads can be used, as seen with both PALM and STORM for example, but these have their own limitations: it is awkward to place the correct amount of beads, to get them to stick so they don't move with respect to the slide, to interrogate them at a wavelength that does not interfere with the sample, and to make them biologically and chemically inert with respect to the sample.
Here we provide a solution that is universal, simple, and immune to sample interference: an inexpensive, easy-to-make, microscope stage mount with a stably built-in ordered, two-dimensional array of fiduciary markers, easily monitored or detected by optical means.
MikroMasch, Inc. (Talinn, Estonia; http://www.spmtips.com/tgz) produces a variety of atomic force microscopy (AFM) tips and calibration gratings. Sun, et al. (Nature Struct and Mol Biol, 17 (4), 485-492, 2010) used such a silicon AFM calibration grating (MikroMasch TGZ03) in a micromolding procedure to make a PMMA microstructure for measuring myosin movement. While Sun's application is unrelated to stage calibration, and his one dimensional (linear grooves and pedestals) microstructures are inapplicable to stage calibration, the same manufacturing processes the can be extended to produce our two-dimensional arrays.